Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites

Recombinant NcPDI(recNcPDI), NcROP2(recNcROP2), and NcMAG1(recNcMAG1) were expressed in Escherichia coli and purified, and evaluated as potential vaccine candidates by employing the C57Bl/6 mouse cerebral infection model. Intraperitoneal application of these proteins suspended in saponin adjuvants lead to protection against disease in 50% and 70% of mice vaccinated with recNcMAG1 and recNcROP2, respectively, while only 20% of mice vaccinated with recNcPDI remained without clinical signs. In contrast, a 90% protection rate was achieved following intra-nasal vaccination with recNcPDI emulsified in cholera toxin. Only 1 mouse vaccinated intra-nasally with recNcMAG1 survived the challenge infection, and protection achieved with intra-nasally applied recNcROP2 was at 60%. Determination of cerebral parasite burdens by real-time PCR showed that these were significantly reduced only in recNcROP2-vaccinated animals (following intraperitoneal and intra-nasal application) and in recNcPDI-vaccinated mice (intra-nasal application only). Quantification of viable tachyzoites in brain tissue of intra-nasally vaccinated mice showed that immunization with recNcPDI resulted in significantly decreased numbers of live parasites. These data show that, besides the nature of the antigen, the protective effect of vaccination also depends largely on the route of antigen delivery. In the case of recNcPDI, the intra-nasal route provides a platform to generate a highly protective immune response.

Phosphocreatine interacts with phospholipids, affects membrane properties and exerts membrane-protective effects

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.

Use of a Th1 Stimulator Adjuvant for Vaccination against Neospora caninum Infection in the Pregnant Mouse Model

Vertical transmission from an infected cow to its fetus accounts for the vast majority of new Neospora caninum infections in cattle. A vaccine composed of a chimeric antigen named recNcMIC3-1-R, based on predicted immunogenic domains of the two microneme proteins NcMIC1 and NcMIC3, the rhoptry protein NcROP2, and emulsified in saponin adjuvants, significantly reduced the cerebral infection in non-pregnant BALB/c mice. Protection was associated with a mixed Th1/Th2-type cytokine response. However, the same vaccine formulation elicited a Th2-type immune response in pregnant mice and did not prevent vertical transmission or disease, neither in dams nor in offspring mice. In this study, an alternative vaccine formulation containing recNcMIC3-1-R emulsified in Freund’s incomplete adjuvant, a stimulator of the cellular immunity, was investigated. No protection against vertical transmission and cerebral infection in the pregnant mice and a very limited protective effect in the non-pregnant mice were observed. The vaccine induced a Th1-type immune response characterized by high IgG2a titres and strong IFN-γ expression, which appeared detrimental to pregnancy.

A multi-tracer study of groundwater origin and transit-time in the aquifers of the Venice region (Italy)

Located in the northeastern region of Italy, the Venetian Plain (VP) is a sedimentary basin containing an extensively exploited groundwater system. The northern part is characterised by a large undifferentiated phreatic aquifer constituted by coarse grain alluvial deposits and recharged by local rainfalls and discharges from the rivers Brenta and Piave. The southern plain is characterised by a series of aquitards and sandy aquifers forming a well-defined artesian multi-aquifer system. In order to determine origins, transit times and mixing proportions of different components in groundwater (GW), a multi tracer study (H, He/He, C, CFC, SF, Kr, Ar, Sr/Sr, O, H, cations, and anions) has been carried out in VP between the rivers Brenta and Piave. The geochemical pattern of GW allows a distinction of the different water origins in the system, in particular based on View the MathML source HCO3-,SO42-,Ca/Mg,NO3-, O, H. A radiogenic Sr signature clearly marks GW originated from the Brenta and Tertiary catchments. End-member analysis and geochemical modelling highlight the existence of a mixing process involving waters recharged from the Brenta and Piave rivers, from the phreatic aquifer and from another GW reservoirs characterised by very low mineralization. Noble gas excesses in respect to atmospheric equilibrium occur in all samples, particularly in the deeper aquifers of the Piave river, but also in phreatic water of the undifferentiated aquifers. He–H ages in the phreatic aquifer and in the shallower level of the multi-aquifer system indicate recharge times in the years 1970–2008. The progression of H–He ages with the distance from the recharge areas together with initial tritium concentration (H + Hetrit) imply an infiltration rate of about 1 km/y and the absence of older components in these GW. SF and Kr data corroborate these conclusions. H − He ages in the deeper artesian aquifers suggest a dilution process with older, tritium free waters. C Fontes–Garnier model ages of the old GW components range from 1 to 12 ka, yielding an apparent GW velocity of about 1–10 m/y. Increase of radiogenic He follows the progression of C ages. Ar, radiogenic He and C tracers yield model-dependent age-ranges in overall good agreement once diffusion of C from aquitards, GW dispersion, lithogenic Ar production, and He production-rate heterogeneities are taken into account. The rate of radiogenic He increase with time, deduced by comparison with C model ages, is however very low compared to other studies. Comparison with C and C data obtained 40 years ago on the same aquifer system shows that exploitation of GW caused a significant loss of the old groundwater reservoir during this time.

Prevention and Immunotherapy of Secondary Murine Alveolar Echinococcosis Employing Recombinant EmP29 Antigen

Alveolar echinococcosis (AE) is caused by infection with the larval stage of the tapeworm Echinococcus multilocularis. An increasing understanding of immunological events that account for the metacestode survival in human and murine AE infection prompted us to undertake explorative experiments tackling the potential of novel preventive and/or immunotherapeutic measures. In this study, the immunoprotective and immunotherapeutic ability of recombinant EmP29 antigen (rEmP29) was assessed in mice that were intraperitoneally infected with E. multilocularis metacestodes. For vaccination, three intraperitoneal injections with 20μg rEmP29 emulsified in saponin adjuvants were applied over 6 weeks. 2 weeks after the last boost, mice were infected, and at 90 days post-infection, rEmP29-vaccinated mice exhibited a median parasite weight that was reduced by 75% and 59% when compared to NaCl- or saponin-treated control mice, respectively. For immunotherapeutical application, the rEmP29 (20μg) vaccine was administered to experimentally infected mice, starting at 1 month post-infection, three times with 2 weeks intervals. Mice undergoing rEmP29 immunotherapy exhibited a median parasite load that was reduced by 53% and 49% when compared to NaCl- and saponin-treated control mice, respectively. Upon analysis of spleen cells, both, vaccination and treatment with rEmP29, resulted in low ratios of Th2/Th1 (IL-4/IFN-γ) cytokine mRNA and low levels of mRNA coding for IL-10 and IL-2. These results suggest that reduction of the immunosuppressive environment takes place in vaccinated as well as immunotreated mice, and a shift towards a Th1 type of immune response may be responsible for the observed increased restriction of parasite growth. The present study provides the first evidence that active immunotherapy may present a sustainable route for the control of AE.

Tracking eolian dust with helium and thorium: Impacts of grain size and provenance

Reconstructions of the deposition rate of windblown mineral dust in ocean sediments offer an important means of tracking past climate changes and of assessing the radiative and biogeochemical impacts of dust in past climates. Dust flux estimates in ocean sediments have commonly been based on the operationally defined lithogenic fraction of sediment samples. More recently, dust fluxes have been estimated from measurements of helium and thorium, as rare isotopes of these elements (He-3 and Th-230) allow estimates of sediment flux, and the dominant isotopes (He-4 and Th-232) are uniquely associated with the lithogenic fraction of marine sediments. In order to improve the fidelity of dust flux reconstructions based on He and Th, we present a survey of He and Th concentrations in sediments from dust source areas in East Asia, Australia and South America. Our data show systematic relationships between He and Th concentrations and grain size, with He concentrations decreasing and Th concentrations increasing with decreasing grain size. We find consistent He and Th concentrations in the fine fraction (<5 μm) of samples from East Asia, Australia and Central South America (Puna-Central West Argentina), with Th concentrations averaging 14 μg/g and He concentrations averaging 2 μcc STP/g. We recommend use of these values for estimating dust fluxes in sediments where dust is dominantly fine-grained, and suggest that previous studies may have systematically overestimated Th-based dust fluxes by 30%. Source areas in Patagonia appear to have lower He and Th contents than other regions, as fine fraction concentrations average 0.8 μcc STP/g and 9 μg/g for 4He and 232Th, respectively. The impact of grain size on lithogenic He and Th concentrations should be taken into account in sediments proximal to dust sources where dust grain size may vary considerably. Our data also have important implications for the hosts of He in long-traveled dust and for the 3He/4He ratio used for terrigenous He in studies of extraterrestrial He in sediments and ice. We also investigate the use of He/Th ratios as a provenance tracer. Our results suggest differences in fine fraction He/Th ratios between East Asia, Australia, central South America and Patagonia, with ratios showing a positive relationship with the geological age of source rocks. He/Th ratios may thus provide useful provenance information, for example allowing separation of Patagonian sources from Puna-Central West Argentina or Australian dust sources. He/Th ratios in open-ocean marine sediments are similar to ratios in the fine fraction of upwind dust source areas. He/Th ratios in mid-latitude South Atlantic sediments suggest that dust in this region primarily derives from the Puna-Central West Argentina region (23–32°S) rather than Patagonia (>38°S). In the equatorial Pacific, He/Th ratios are much lower than in extratropical Pacific sediments or potential source areas measured as a part of this study (East Asia, South America, Australia) for reasons that are at present unclear, complicating their use as provenance tracers in this region.

N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response

Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies.